Hyclone

Bladder cancer cell lines (5637, T24 and UM-UC-3) and an immortalized urothelial cell line (SV-HUC-1) were obtained from China Infrastructure of Cell Line Resources, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. All cells were cultured at 37°C with 5% CO₂ and 95% O₂. SV-HUC-1 cells were cultured in a 1:1 mixture of DMEM and Ham's F12 medium (both HyClone; Cytiva). 5637, T24 and UM-UC-3 cells were cultured in RPMI-1640 medium (HyClone; Cytiva) supplemented with 10% FBS (HyClone; Cytiva), 100 U/ml penicillin G and 100 µg/ml streptomycin (Lonza Group, Ltd.).

All animal procedures were approved by the Animal Ethics Committee of Zaozhuang Maternal and Child Health Care Hospital. In total, 84 C57BL/6 mice (weight, 10.3±0.9 g; age, 21 days; total number, 84; immature females) were from Shanghai SLAC Laboratory Animal Co., Ltd. The mice were housed in a temperature (22-24˚C) and humidity (50-60%) controlled environment with a 12-h light/dark cycle and given ad libitum access to food and water. The mice were intraperitoneally injected with 8 IU pregnant mare serum gonadotrophin (Ningbo Sansheng Pharmaceutical Industry Co., Ltd.). At 47 h post-injection, mice were sacrificed by cervical dislocation, the ovaries were collected and the surrounding tissues removed. GCs were isolated from the ovaries by puncturing the follicles with 26-gauge needles, followed by centrifugation at 300 x g for 10 min at 4˚C. GCs were cultured in DMEM/F12 (HyClone; Cytiva) supplemented with 10% FBS (HyClone; Cytiva) and 1% penicillin/streptomycin (HyClone; Cytiva) at 37˚C with 5% CO₂. At 1 day post-transduction, cells were incubated with 10 µM testosterone (Sigma-Aldrich; Merck KGaA) or vehicle (0.1% DMSO) for 24 h at 37˚C to induce apoptosis. In addition, cells were treatment with 10 µM testosterone together with 1 µM flutamide (Sigma-Aldrich; Merck KGaA) at 37˚C for 24 h.

The human HNSCC cell lines, Tu686 and FaDu (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences), were used in the present study. HuLa-PC (CRL-3342; ATCC), a human normal laryngeal cell line, was also used in the present study. Tu686 was cultured using RPMI-1640 medium (HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin. FaDu and HuLa-PC was cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone; Cytiva) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells were placed in an incubator with 5% CO₂ at 37°C. AMC-HN8 cells were a kind gift from Professor Sang Yoon Kim of Samsung Medical Center (Seoul, Korea). Tu212 and M4E cell lines were obtained from Central South University (Changsa, China). AMC-HN8, Tu212 and M4E were cultured using RPMI-1640 medium (HyClone; Cytiva) supplemented with 10% and 1% penicillin-streptomycin, and were placed in an incubator with 5% CO₂ at 37°C. For the mRNA stability assay, transfected HNSCC cells were treated with Actinomycin D (5 µg/ml; cat. no. S8964; Selleck Chemicals) at 37°C for 0, 3 and 6 h prior to RNA isolation.

The human colorectal adenocarcinoma cell line, SW480, was purchased from the American Type Culture Collection (CCL-228™) and cultured in DMEM/high glucose medium (HyClone; Cytiva; cat. no. SH30022.01B) containing 10% fetal bovine serum (HyClone; Cytiva; cat. no. SH30087.01) at 37°C in a 5% CO₂ incubator. Sulforaphane (Abcam; cat. no. ab141970) and sulforaphene (Abcam; cat. no. ab141972) were purchased from Abcam (purity >98%), solubilized in double-distilled water and diluted to 25 µmol/l in culture medium. The RNA TRIzol^® extraction kit was purchased from Invitrogen, Thermo Fisher Scientific, Inc.. The GN-genechip Clariom™ S Array next-generation sequencing (NGS) chip was purchased from Affymetrix, Thermo Fisher Scientific, Inc. (cat. no. 902927; human).

Eca109, KYSE30, KYSE150, TE-1 and KYSE510 cells were purchased from Wuhan University, China, and maintained in RPMI-1640 medium (HyClone; Cytiva) enriched with 10% FBS (Shanghai VivaCell Biosciences, Ltd.) and penicillin-streptomycin (HyClone; Cytiva) in a humidified atmosphere containing 5% CO₂ at 37°C.

A frozen aliquot of the third passage of NSPCs was donated by the Department of Neurosurgery and Key Laboratory of Neurotrauma, Southwest Hospital, Army Military Medical University (Chongqing, China) and was used for further experiments (20 (link)). As previously described, primary NSPCs were isolated from adult male C57BL/6 mice (age, 6-8 weeks; weight, 18-22 g) (20 (link)). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO₂. Cells in the logarithmic growth phase were subjected to trypsinization and subculture with 0.25% trypsin (HyClone; Cytiva). The OGD model was established as previously described (20 (link)). Cells were incubated with glucose-free and FBS-free Earle's BSS buffer (Thermo Fisher Scientific, Inc.) at 37˚C for 8 h with 94% N₂, 5% CO₂ and 1% O₂. Different concentrations of ART (0.25, 0.5, 1, 2 and 4 µmol/l) and IL-6 (2 ng/ml) were added to medium to treat NSPCs for 48 h after ODG.